T cells recognize antigens only when bound by antigen presenting molecules. Peptides generated in the cytosol are transported into the ER where they bind MHC class I molecules, while MHC class II molecules are directed to endocytic compartments were they bind peptides. Thus antigen-binding is essentially determined by the differential trafficking and loading compartments of MHC class I and II molecules. Now, it is recognized that lipid antigens also can be recognized by T cells when presented by CD1 molecules. Like MHC molecules, trafficking determines where each of the CD1 isoforms binds antigens. CD1a traffics mainly through early endosomes and the early recycling compartment, while CD1b and CD1d traffic instead to late endosomes and lysosomes. Antigen binding occurs when CD1 molecules and lipid antigens intersect in these endosomal compartments. Since CD1 molecules bind lipid antigens in distinct endocytic compartments, they must then recycle or travel outbound to the cells surface. We and others have defined many of the key features of CD1 assembly in the ER, delivery to the cell surface and then internalization to localize in endosomes where lipid antigens are bound. Here, we developed a custom shRNA trafficking library and applied it in a screen to identify molecular mediators of CD1d recycling through lysosomes that is essential to deliver these molecules from the endosome compartments where they bind antigens to the cell surface where they can be recognized by T cells. We found, for the first time, that novel members of the Ras family of small GTPases, called Arl proteins, in particular Arl8, are essential regulators of lysosomal trafficking that is important in antigen presentation and phago-lysosome fusion. First, we will examine how lysosomal trafficking is blocked resulting in lysosome clustering in Arl8b shRNA knockdown or dominant- negative mutant expressing cells (Aim 1). Then, we will determine the role of Arl8b in the trafficking and presentation of antigens by CD1b and CD1d molecules that must traffic to lysosomes to acquire their lipid antigens and then deliver them to the cell surface for recognition (Aim 2). Beyond these CD1 antigen presenting molecules, MHC class II molecules also must traffic to and acquire antigens in lysosomes before delivering them to the cells surface. We will determine how Arl8b blocks MHC class II antigen presentation and their trafficking to the surface in the physiologically important process of dendritic cell maturation (Aim 3). We will then examine the role of Arl8b in the lysosomal trafficking necessary for delivery of its content to phagosomes in phago-lysosome fusion (Aim 4). Finally, we will identify molecules that bind Arl8b (Arl8 effectors) that enable it to mediate lysosomal trafficking through linkage to tethers, Rabs and the microtubular cytoskeleton (Aim 5). These studies will provide new insights into the biology of lysosomal trafficking and reveal how it impacts antigen-presenting molecules and other lysosomal contents that travel out from their endocytic locations to reach critical destinations at the cell surface (for T cell recognition) or to phagosomes (for microbial killing). PUBLIC HEALTH RELEVANCE: Antigen presentation by CD1 and MHC molecules is responsible for eliciting adaptive immunity. Here we determine how CD1 and MHC molecules that have bound microbial antigens are able to travel to the cell surface of antigen presenting cells to simulate T lymphocyte immune responses. Understanding how this pathway of antigen presentation works will make it possible to optimize it to generate effective vaccines for a wide variety of microbial infections. Further, we determine what regulates the process of how cells kill microbes by fusing the vesicles containing the organisms with vesicles that contain factors that can destroy the infections agents.